Analytical test device and method of use

ABSTRACT

The present invention provides an analytical test device for conducting assays of biological fluids. Methods for carrying out the assays with the disclosed analytical test device are also provided.

FIELD OF THE INVENTION

[0001] This invention relates to a novel analytical test device foranalysis of biological fluids such as urine. The invention furtherrelates to methods for detecting analytes in fluids using the testdevice.

BACKGROUND INFORMATION

[0002] The sampling and testing of biological fluids such as urine forthe presence of analytes provide important information regarding varioushealth-related matters, including pregnancy and conception.

[0003] In recent years, test devices have undergone nearly continuousrefinement in an effort to simplify and speed the process of detectingselected ligand in fluids. As a result of this work, current testdevices use an immunoassay for determining pregnancy or conception. Inthese devices a reagent, such as an antibody, specifically reacts withan analyte to form a complex, which can usually be detected by theunaided eye.

[0004] Current pregnancy test devices assay for hormones associated withpregnancy, such as, for example, chorionic gonadotrophin (hereinafter“hCG”) since the presence of hCG in urine is usually an indicator that awoman is pregnant. Such test devices obtain qualitative resultsindicating either the presence or absence of hCG. Typically, a pregnancyimmunoassay contains an antibody directed against hCG. The reactioncomplex can then be viewed by the user.

[0005] Conception test devices also assay for hormones associated withthe ovarian cycle, such as, for example, luteinizing hormone(hereinafter “LH”). LH is present normally in urine but itsconcentration increases markedly during ovulation, the time at which awoman is most likely to conceive. Thus, the probability that a woman canconceive a child varies directly with LH concentration. Such testdevices obtain semi-quantitative results regarding the relativeconcentration of LH in the urine. Typically, a conception immunoassaycontains an antibody directed against LH and a separate detectionantibody.

[0006] In known devices, the fluid to be tested wicks up through anabsorbent membrane that is in fluid flow contact with the reagents thatdetect an analyte in a fluid. A major problem with this type of deviceis that as the fluid progresses through the membrane, the front edge ofthe fluid is uneven. The uneven fluid front will cause the results to besmeared, produce false negatives and make the results difficult todetect. These artifacts are called “leading edge effects.” An unevenfluid front can arise for a variety of reasons, for example, because thesample is not uniformly applied to the absorbent membrane.

[0007] Therefore, a need exists for a device that does not produce anuneven fluid front and associated leading edge effects. The presentinvention provides a device that eliminates the uneven fluid frontproblem and related methods of using such a device.

SUMMARY OF THE INVENTION

[0008] The present invention provides an analytical test device foranalyzing biological fluids, for example, urine. The device has a topwith one or more display ports and, optionally, a receiving port and avertical bar. The device also has a bottom and, optionally, a receivingport, an upper plane, a slope and a lower plane. The device further hasa strip made up of an absorbent membrane, a reaction membrane with oneor more reagents that form a reaction complex with an analyte in a fluidsample and, optionally, a collecting pad. The device also has anabsorbent sample pad in a container and a stopping means.

[0009] The absorbent membrane, the reaction membrane and the collectingpad, when present, are in fluid flow contact with one another. The stripis placed on the bottom such that the reaction membrane is visiblethrough the display port and the absorbent membrane is toward thereceiving port. The top or the bottom has the receiving port or togetherthe top and the bottom form the receiving port and in the assembled topand bottom the absorbent sample pad is slidably insertable through thereceiving port until stopped by the stopping means, which places theabsorbent sample pad in fluid flow contact with the absorbent membrane.

[0010] In one embodiment, the analytical test device also has the topwith the optional vertical bar. The vertical bar deflects the absorbentmembrane to follow the contour of the slope. The bottom has the optionallower plane, upper plane and slope and also a stopping means.

[0011] The present invention also provides methods for detecting ananalyte in a fluid sample, comprising adding fluid sample to theabsorbent sample pad, inserting the absorbent sample pad into thereceiving port until the absorbent sample pad is stopped by the stoppingmeans and detecting the analyte by observing the reaction complexthrough the display port. In one method the reaction complex is visible.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 is a perspective view of the analytical test devicecontaining an absorbent sample pad in a container.

[0013]FIG. 2 is an exploded view showing the components of theanalytical test device of FIG. 1.

[0014]FIG. 3 is a perspective view of an embodiment of an analyticaltest device with an absorbent sample pad inside a container.

[0015]FIG. 4 is an exploded view showing the components of theanalytical test device of FIG. 3.

[0016]FIG. 5 is an enlarged sectional view along line 3-3 of theanalytical test device of FIG. 3 showing that the absorbent sample padis not in fluid flow contact with the absorbent membrane.

[0017]FIG. 6 is an enlarged sectional view along 3-3 of the analyticaltest device of FIG. 3 showing that the absorbent sample pad is in fluidflow contact with the absorbent membrane.

[0018]FIG. 7 is a perspective view of an embodiment of an analyticaltest device containing a slidably insertable absorbent sample pad in acontainer.

[0019]FIG. 8 is an exploded view showing the components of theanalytical test device of FIG. 7.

[0020]FIG. 9 is an enlarged sectional view along line 3-3 of theanalytical test device of FIG. 7 showing initial placement of theabsorbent sample pad not in fluid flow contact with the absorbentmembrane.

[0021]FIG. 10 is an enlarged sectional view along line 3-3 of theanalytical test device of FIG. 7 showing placement of the absorbentsample pad in fluid flow contact with the absorbent membrane.

[0022]FIG. 11 is a perspective view of the absorbent and reactionmembranes of the analytical test device employing a sandwich assaysystem showing a positive assay result.

[0023]FIG. 12 is a perspective view of the absorbent and reactionmembranes of the analytical test device employing a sandwich assaysystem showing a negative assay result.

[0024]FIG. 13 is a perspective view of the absorbent and reactionmembranes of the analytical test device employing a sandwich assaysystem showing an inconclusive assay result.

[0025]FIG. 14 is a perspective view of the absorbent and reactionmembranes of an embodiment of the analytical test device employing acompetitive assay system showing a positive assay result.

[0026]FIG. 15 is a perspective view of the absorbent and reactionmembranes of an embodiment of the analytical test device employing acompetitive assay system showing a negative assay result.

DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention is directed to an analytical test devicethat eliminates the uneven fluid front and leading edge effectsassociated with prior devices. The analytical test device ensures thatfluid sample comes into fluid flow contact with the membrane thatcontains the reagents to detect the analyte, evenly and at the sameheight across the membrane, thereby producing an even fluid front.

[0028] Fluid sample is collected in an absorbent sample pad that isinitially not in fluid flow contact with the absorbent membrane. Thenthe absorbent sample pad is contacted to the absorbent membrane so thatthe fluid sample comes into fluid flow contact with the absorbentmembrane. The edge of the absorbent sample pad contacts the absorbentmembrane orthogonal to the absorbent membrane, ensuring that the fluidsample will contact the absorbent membrane evenly and at the same heightacross the membrane.

[0029] A further advantage of the analytical test device is that theuser can control when the assay starts. Thus, if desired, the user canadd more than one fluid sample to the absorbent sample pad before theassay starts. This feature is especially important where the user onlywishes to know whether an analyte is present in more than one fluidsample. In prior devices, once the fluid sample is added, the assayproceeds and the reagents are depleted before a second sample can beadded. Thus, the user is not given the time to test additional samples.

[0030] The analytical test device of the present invention includes atop and a bottom. The top has a display port that allows the user to seethe results. Preferably, the top will have a first display port and asecond display port that are in close proximity for easy comparison ofresults. The bottom has a receiving port for an absorbent sample pad.Inside the device is a strip formed by an absorbent membrane, a reactionmembrane and, optionally, a collecting pad. The absorbent membrane andthe reaction membrane contain the reagents to detect an analyte in thefluid sample. When the top is placed on the bottom in the assembleddevice, the absorbent membrane will be positioned on the bottom towardthe receiving port and the reaction membrane will be positioned on thebottom underneath the display port.

[0031] The absorbent sample pad is held in a container. In oneembodiment, the absorbent sample pad will be protruding from thecontainer. Fluid sample is deposited on the absorbent sample pad and, ifdesired, more than one fluid sample can be applied or contacted with theabsorbent sample pad before the test is allowed to proceed. At thispoint the absorbent sample pad is not in fluid flow contact with thestrip. The absorbent sample pad is then inserted into the receiving portuntil the absorbent sample pad is in fluid flow contact with theabsorbent membrane. The analytical test device also has a stopping meansthat prevents the absorbent sample pad from reaching far enough into theassembled top and bottom to by-pass the absorbent membrane and contactthe reaction membrane.

[0032] The bottom of the analytical test device of the present inventioncan also be divided into an upper plane and a lower plane by a slope. Asused herein, the term “slope” refers to a surface or plane of the bottomof the device that is at such an angle in relation to the other planesof the bottom that it slows the rate of fluid sample wicking through theabsorbent membrane. Thus, the slope prevents an excess amount of fluidsample migrating through the absorbent membrane, which would otherwiseand compromise the assay.

[0033] When the slope is present, the absorbent membrane is along thelower plane and the slope and the reaction membrane is on the upperplane. Also, the analytical test device has a vertical bar extendingdownward from the top. As used herein, the term “vertical bar” refers toa part of the top of the device extending downward from the top at apoint just in front of the slope that holds or deflects the absorbentmembrane to follow the contour of the slope. Optionally, the verticalbar can extend downward far enough to only allow fluid sample to proceedfurther into the device by wicking through the absorbent membrane.

[0034] In one embodiment, the absorbent sample pad is inside a containerthat can be slidably inserted through the receiving port until it comesinto fluid flow contact with the absorbent membrane. In a furtherembodiment, the absorbent sample pad is in a container separate from theassembled top and bottom. The absorbent sample pad protrudes from thecontainer and can be inserted into the receiving port. In anotherembodiment, the absorbent sample pad is in a container that is disposedbetween the assembled top and bottom of the analytical test device, andpartially protruding therefrom. A knob attached to the container allowsthe user to slide the container inside the device until the stoppingmeans stops the container and the absorbent sample pad contacts theabsorbent membrane.

[0035] An amount of fluid sample, such as urine, which is suspected ofcontaining, for example hCG, is applied by dropping or pouring (from apipette or other container) the sample onto the absorbent sample pad.Alternatively, in one embodiment, the absorbent sample pad can be dippedinto a fluid sample. In addition, the absorbent sample pad may be wettedby carefully holding in a stream of urine. The absorbent sample pad isthen contacted with the absorbent membrane by inserting the absorbentsample pad through the receiving port until the container is stopped bythe stopping means.

[0036] The fluid sample then proceeds by capillary action (i.e.,wicking) through the absorbent membrane. Bound evenly and at the sameheight extending from side to side of the absorbent membrane, at adownstream point spatially separated from the point where the absorbentsample pad contacts the absorbent membrane and downstream from thevertical bar is a diffusively bound labeled reagent. The spatialseparation ensures that the fluid sample wicks up through the absorbentmembrane to contact the diffusively bound labeled reagent. Thus, thediffusively bound labeled reagent stays within the absorbent membraneand does not migrate into the fluid sample, which would otherwisedestroy the performance of the assay.

[0037] If analyte is present in the fluid sample it will complex withthe diffusively bound labeled reagent. Fluid sample containing eitherdiffusively bound labeled reagent alone or a diffusively bound labeledreagent/analyte complex (i.e., “first complex”) will move by wicking upto a “test region” in the reaction membrane. Bound evenly and at thesame height extending from side to side of the reaction membrane in thetest region is a non-diffusively bound reagent capable of complexing thefirst complex. The non-diffusively bound reagent may be non-diffusivelybound to the membrane by means known in the art, including covalentbonding or attachment to an insoluble protein-coated surface (see, forexample, U.S. Pat. No. 4,200,690, which is incorporated herein byreference). Preferably, the non-diffusively bound reagent will be boundin a bar shape extending from side to side of the reaction membrane in amanner similar to the attachment of the diffusively bound labeledreagent to the absorbent membrane.

[0038] If a first complex is present in the fluid sample, the analyteportion of the first complex will bind to the non-diffusively boundreagent creating a detectable signal, preferably a visible straight lineor bar extending from side to side of the reaction membrane at the firstdisplay port. As a control, fluid sample will continue its migrationpast the first display port to a “control region” portion of thereaction membrane.

[0039] The control region is so called because bound evenly and at thesame height extending from side to side of the reaction membrane is anon-diffusively bound control reagent. The diffusively bound labeledreagent, complexed with analyte or not, will bind to the non-diffusivelybound control reagent present in the control region. This binding willcreate a detectable signal, preferably a visible straight line or barextending from side to side of the reaction membrane at the seconddisplay port. For ease of comparison, the shape and orientation of thenon-diffusively bound control reagent should be similar to the shape andorientation of the non-diffusively bound reagent. In addition, thenon-diffusively bound control reagent may be non-diffusively bound inthe manner similar to that of the non-diffusively bound reagent,described above. When the top is aligned with the bottom in theassembled device the diffusively bound labeled reagent will be bound soas to be spatially separated from the point where the absorbent samplepad contacts the absorbent membrane, the non-diffusively bound reagentwill be bound beneath first display port and the non-diffusively boundcontrol reagent will be bound beneath second display port.

[0040] The results can then be interpreted. If a signal appears at thefirst display port and a signal appears at the second display port, thetest is positive for the presence of analyte. If there is no signaldetectable at the first display port compared to the signal at thesecond display port, the test is negative for the presence of analyte.If there is no signal at the second display port, the user is alertedthat the test results are compromised and that the test should berepeated.

[0041] Alternatively, the analytical test device can employ acompetitive assay system. A diffusively bound labeled reagent capable ofcompeting with the analyte for a non-diffusively bound reagent isapplied evenly and at the same height extending from side to side of theabsorbent membrane. It is applied at a downstream point spatiallyseparated from the point where the absorbent sample pad contacts theabsorbent membrane and downstream from the vertical bar. The diffusivelybound labeled reagent here does not complex with the analyte. Anon-diffusively bound reagent that can bind either the analyte or thediffusively bound reagent is applied evenly and at the same heightextending from side to side of the reaction membrane at a point beneaththe display port. In this embodiment of the analytical test device, thetop need only have one display port, as no comparison is required.However, if desired, a second display port may be incorporated todemonstrate the integrity of the reagents and device. In thisembodiment, the presence of a visible signal, such as a straight line orbar across the reaction membrane in the display port is a validation ofthe assay and indicates that no analyte is present in the fluid sample.No signal in the display port indicates the presence of analyte in thefluid sample.

[0042] The analytical test device allows the assay to be performed insimple steps. The user only has to apply the fluid sample, slide theabsorbent sample pad into the receiving port to contact the absorbentsample pad with the absorbent membrane and then observe the results.

[0043] The analytical test device is assembled by placing the absorbentmembrane and the reaction membrane on the bottom and then the top isthen placed on the bottom to ensure a tight fit. One skilled in the artwould understand that any suitable means to ensure a tight fit betweenthe top and bottom can be used. For example, the top can be fitted onthe bottom with snaps or glue. In addition, once the absorbent samplepad is inserted into the receiving port, the device becomessubstantially fluid-tight and evaporation or leakage of the fluid sampleis minimized. To further prevent such leakage, the top and bottom arepreferably made of a nonporous plastic, such as the commerciallyavailable plastic “ABS” supplied by the Monsanto Company of St. Louis,Mo.

[0044] Several features of the analytical test device assist in avoidingcompromised results. First, simultaneous migration of an excessiveamount of fluid sample is limited by requiring the fluid sample toascend the slope from the absorbent membrane to the reaction membrane.Second, the spatial separation of the diffusively bound labeled reagenton the absorbent membrane from the point at which the fluid sample firstcontacts the absorbent membrane ensures that the assay is notcompromised by reagent leaking out into the fluid sample. Third, theimpact of leading edge effects caused by depletion of bound reagents orlabels are minimized by physically separating the test and controlregions, by applying the fluid sample evenly and at the same heightacross the absorbent membrane and by using the presence of a reaction,rather than the appearance of a given shape (such as a “+” or a “−”) tosignify a positive or negative result. In addition, the entire processis simplified by having the label bound to a substrate placed within theanalytical test device, thus avoiding any need for the user to add areagent or mix it with fluid sample.

[0045] As described above, the present invention is also ideally suitedto the application of more than one fluid sample to a single analyticaltest device. In prior devices, such as the one described in May et al.,U.S. Pat. No. 5,602,040, issued Feb. 11, 1997, the device (hereinafterthe '040 device) is typically held in a stream of urine. The protrudingsample aperture in the '040 device is in contact with the dry porouscarrier inside the hollow casing of the device. Thus, the test proceedsimmediately upon application of the sample. Therefore, the '040 deviceis suited for the testing of only one sample at a time because once thefirst sample is applied, the test proceeds, thereby depleting thereagents.

[0046] Moreover, since the user of the '040 device typically holds theprotruding bibulous member in a urine stream, the sample is unevenlyapplied to the membrane. Thus, the '040 device suffers from leading edgeeffects created by an uneven solvent front along the membrane.Accordingly, reagents and bound labels could be depleted and the resultscould be misleading. In contrast, the user of the present analyticaltest device is assured that the solvent front formed by the fluid samplewill be uniform since the fluid sample is applied evenly and at the sameheight across the absorbent membrane. Therefore, the present analyticaltest device provides more reliable results.

[0047] As used herein, the terms “ligand” and “control reagent” are usedinterchangeably and refer to a molecule to which another molecule willbind. A ligand or control reagent can be human, animal or plant inorigin. For purposes of this invention, these may include LH, hCG orother naturally occurring biological control reagents in serum, plasma,urine or other fluids and will preferably include analytes.Specifically, it will be appreciated by those skilled in the art thatthe control reagent or analyte may be a protein, peptide, amino acid,nucleic acid, sugar, hormone, steroid, vitamin, toxin in the samplefluid, pathogenic microorganism and metabolites from analogs of suchanalytes, or antibodies to these substances. They may also includehormones, haptens, immunoglobulin, polynucleotides, drugs and infectiousdisease agents (bacterial or viral) such as Streptoccus, Neisseria,Chlamydia, Gonorrhea and HIV.

[0048] As used herein, the term “reagent” refers to receptor moleculeswhich bind to a control reagent. Reagents may, in this context, includeany naturally occurring or synthetic biological receptor and willpreferably include antibodies. The antibodies may be polyclonal ormonoclonal. For simplicity, the terms antibody and analyte will be usedinterchangeably with reagent and control reagent, respectively,throughout this disclosure. It will be appreciated by those skilled inthe art, however, that the invention is not limited to use with analytesand antibodies.

[0049] Ligand-reagent pairs useful in the present invention includespecific binding pairs such as antigens and antibodies, or fragments ofantibodies, both polyclonal and monoclonal, lectins and carbohydrates,hormones and hormone receptors, enzymes and enzyme substrates, biotinand avidin, vitamins and vitamin binding proteins, complementarypolynucleotide sequences, drugs and receptors, enzymes and inhibitors,apoproteins and cofactors, growth factors and receptors, and the like.Biotin and avidin derivatives may also be used, including biotinanalogs/avidin, biotin/streptavidin, and biotin analogs/streptavidin.Members of the complex may be “engineered,” that is, made by syntheticmeans. Such techniques are well known in the art, and include techniquesfor chimeric and humanized antibodies and fragments thereof, syntheticpeptides, and synthetic RNA and DNA oligonucleotides.

[0050] Any known reagent can be used in any known format such as, forexample, sandwich and competitive binding formats, to specificallydetect an analyte in a fluid sample. Examples of such reagents are thosedisclosed in: H. J. Friesen, U.S. Pat. No. 4,861,711, issued Aug. 29,1989; J. Bunting, U.S. Pat. No. 4,271,140, issued Jun. 2, 1981; May etal., U.S. Pat. No. 5,622,871, issued Apr. 22, 1997; May et al., U.S.Pat. No. 5,656,503, issued Aug. 12, 1997; May et al., U.S. Pat. No.5,602,040, issued Feb. 11, 1997; and R. Rosenstein, U.S. Pat. No.5,591,645, issued Jan. 7, 1997 (each of which is incorporated byreference herein). Such reagents can form a detectable complex with suchcontrol reagents as listed above.

[0051] Preferred reagents include antibodies to a hormone or infectiousdisease agent. Preferred antibodies include anti-hCG antibodies andanti-human LH antibodies, especially of the IgG class, and even more somurine monoclonal antibodies and especially those that have beenaffinity purified. One skilled in the art would recognize, however, thatpolyclonal antibodies can also be used in the present invention.

[0052] By the term “non-diffusively bound” is meant covalent ornon-covalent attachment to the absorbent membrane or reaction membranesuch that the advancing fluid sample does not cause movement of thenon-diffusively bound reagent or non-diffusively bound control reagentfrom the place it is applied on these membranes. Conversely, by the term“diffusively bound” is meant placement on the absorbent membrane orreaction membrane such that the advancing fluid sample does causemovement of the diffusively bound labeled reagent from the place it isapplied on these membranes.

[0053] As used herein, the term “fluid sample” refers to a materialsuspected of containing an analyte. The fluid sample can be useddirectly as obtained, for example, from any biological source. The fluidsample can also be obtained from an organism and the relevant portionextracted or dissolved into a solution. For example, the fluid samplecan be a physiological fluid, such as, for example, saliva, ocular lensfluid, cerebral spinal fluid, sweat, blood, pus, mucus, serum, urine,milk, ascites fluid, synovial fluid, peritoneal fluid, amniotic fluid,and the like. In addition, the fluid sample fluid can be extracted fromthroat swabs, feces, or from other biological samples.

[0054] As used herein, the term “label” refers to a molecule thatdirectly or indirectly mediates the production of a signal (such as acolor change) which is used in assay procedures to indicate the presenceor absence of analyte in a fluid sample. Labels may include enzymes,fluorescent molecules and will preferably include metal sols. Labelsinclude colored latex spheres and colloidal metals. Labels include thosedisclosed by D. Yost et al., U.S. Pat. No. 4,954,452, issued Sep. 4,1990; J. Leuvering, U.S. Pat. No. 4,313,734, issued Feb. 2, 1982; P.Tarcha et al., U.S. Pat. No. 5,252,459, issued Oct. 12, 1993; T. Gribnauet al., U.S. Pat. No. 4,373,932, issued Feb. 15, 1983; and R. Campbell,U.S. Pat. No. 4,703,013, issued Oct. 27, 1987 (each of which isincorporated by reference herein).

[0055] Alternatively, the label can be colored latex particles (seeCampbell, U.S. Pat. No. 4,703,017, issued Oct. 27, 1987, which isincorporated by reference herein) or can be an enzyme that has reactedwith a colorless substrate to give a colored product and isencapsulated, for example, in a liposome (see E. Soini, U.S. Pat. No.5,518,883, issued May 21, 1996, which is incorporated herein byreference). The label may also be an inducible property of theparticles, such as colorable latex particles (see Gribnau et al., U.S.Pat. No. 4,373,932, issued Feb. 15, 1983, and de Jaeger et al., U.S.Pat. No. 4,837,168, issued Jun. 6, 1989, both of which are incorporatedby reference herein).

[0056] Alternatively, the label can be fluorescent molecules, such asthe rhodamine, fluorescein, or umbelliferone series, employed bythemselves or with a quencher molecule (see, for example, Ullman et al.,U.S. Pat. No. 3,996,345, issued Dec. 7, 1976 and Tom et al., U.S. Pat.No. 4,366,241, issued Dec. 28, 1982, both of which are incorporatedherein by reference). Chemiluminescent molecules, such as luminol,luciferin, lucigenin, or oxalyl chloride can be used as a signal means(see, for example, Maier, U.S. Pat. No. 4,104,029, issued Aug. 1, 1978,which is incorporated herein by reference). Finally, enzymatic systemsthat react with a colorless substrate to give a colored product, such ashorseradish peroxidase, alkaline phosphatase, indoxyl phosphate andaminoethylcarbazole may be used as labels.

[0057] Diffusively bound complexed antibodies can be impregnated intothe assay and within the absorbent membrane. The diffusively boundcomplexed antibodies are located upstream from a zone on the reactionmembrane of the assay that contains immobilized antibodies. Thediffusively bound label complexed antibodies bind to the analyte and arecarried to the zone containing the immobilized antibodies where asandwich antibody-hormone complex is formed and detected. However, ifdesired, both the diffusively bound and non-diffusively bound antibodiesmay be disposed on the same membrane.

[0058] In one embodiment, the diffusively bound labeled reagent will belabeled according to means known in the art. For purposes of producing aclearly visible reaction, labels of metal sols are preferred, withlabels of colloidal gold or selenium being most preferred. An example ofa suitable product is colloidal gold. These colloidal metals willproduce colored reactions without addition of further reagents. However,fluorescent compounds (such as fluorescein and phycobiliprotein) andenzymes (such as those identified in U.S. Pat. No. 4,275,149, which isincorporated by reference herein), may also be used. To maximize contactof analyte with labeled reagent, the latter should be bound to themembrane across its face; i.e., from one side to the other. In addition,the labeled reagent should be bound to the membrane so that it isspatially separated from the absorbent sample pad when the absorbentsample pad is contacted with the absorbent membrane (see bar 125 shownin phantom on FIGS. 9, 10, 11, 12 and 13).

[0059] As used herein, the term “metal label” includes labels of metalsols; i.e., metal or metal compounds such as metal oxides, metalhydroxides, metal salts or polymer nuclei coated with a metal or metalcompound. These metal labels may include dry forms of any of theabove-named metals or metal compounds, and will preferably includecolloidal gold in dry form. For example, the metal label can be composedof a metal sol, a selenium sol or a carbon sol (see, for example,Leuvering et al., U.S. Pat. No. 4,313,734, issued Feb. 2, 1982; Moremanset al., U.S. Pat. No. 4,775,636, issued Oct. 4, 1988; Yost et al., U.S.Pat. No. 4,954,452, issued Sep. 4, 1990; and Kang et al., U.S. Pat. No.5,559,041, issued Sep. 24, 1996, each of which is incorporated byreference herein).

[0060] Depending on the context in which it is used, “reaction complex”or “complex” shall mean an a complex of analyte and diffusively boundlabeled reagent complex first produced in the assay (“first complex”), acomplex of the first complex and the non-diffusively bound reagentproduced second in the assay (“second complex”), or the second complexand the non-diffusively bound control reagent produced third in theassay (“third complex”). In another embodiment of the analytical testdevice, “complex” shall mean a complex of analyte and non-diffusivelybound reagent or a complex of the diffusively bound labeled reagent andnon-diffusively bound reagent. The complex can be made up of aligand-reagent pair that has spatial and/or polar features which permitit to bind specifically to each other. By “first member” of the complexis meant the member bound, covalently or non-covalently, at least to asub-population of the particles. The “second member” of a complex refersto the corresponding binding member of the pair non-diffusively bound inthe control region.

[0061] The term “display port” refers to any means whereby visual accessto the reaction membrane can be gained. In one embodiment, a displayport will be an aperture on the top positioned over the reactionmembrane. The term also encompasses all or any part of the top that ismade of a clear or transparent material so that the results can be seen.Thus, if the entire top is made of clear or transparent material, thenthe entire top can be referred to as the display port. In addition,there may be a clear or transparent membrane placed between the reactionmembrane and the aperture of the display port so as to preventevaporation of fluid sample, to prevent fluid sample or other fluid fromentering the display port, or to prevent disruption of the membranes bytouching, all of which can affect the performance of the assay.

[0062] The term “absorbent sample pad” refers to any material capable ofcontaining the fluid sample and when contacted with the absorbentmembrane produces an even fluid front along the absorbent membrane. Forexample, the edge of the absorbent sample pad that comes into contactwith the absorbent membrane should be orthogonal to the absorbentmembrane, ensuring that fluid sample will be applied evenly and at thesame height across the membrane, thereby producing an even fluid front.The absorbent sample pad can be made from any bibulous, porous orfibrous material capable of absorbing fluid rapidly. The porosity of thematerial can be unidirectional or multidirectional. Porous plasticmaterials, such a polypropylene, polyethylene (preferably of highmolecular weight), polyvinylidene fluoride, ethylene vinylacetate,acrylonitrile and polytetrafluoroethylene can be used. It can beadvantageous to pre-treat the pad with a surface-active agent duringmanufacture to reduce any inherent hydrophobicity in the pad and,therefore, enhance its ability to take up and deliver a fluid samplerapidly and efficiently. The absorbent sample pad can also be made frompaper or other cellulosic materials, such as nitro-cellulose. Preferablythe material comprising the absorbent sample pad should be chosen suchthat the pad can be saturated with fluid sample within a matter ofseconds. Also preferably, the material remains robust when moist, andfor this reason, paper and similar materials are less preferred. Inaddition, by preferably providing a tight fit between the absorbentsample pad and the assembled top and bottom, the application of fluidsample to the absorbent sample pad will not result in fluid sampleentering the device directly and by-passing the absorbent sample pad.

[0063] Also, the absorbent sample pad of the present invention ismovable or “slidably insertable.” As used herein, the term “slidablyinsertable” refers to the ability of the absorbent sample pad to bemoved inside the device. In one embodiment of the device, the absorbentsample pad is disposed between the top and the bottom and can be slidinside the assembled top and bottom by the user (see FIG. 1). Theabsorbent sample pad will be in an container disposed between the topand bottom in the assembled device. The container is preferably made outof the same non-porous material as the top and bottom. In anotherembodiment of the device, the absorbent sample pad will be initiallyseparate from the assembled top and bottom but insertable into thereceiving port. The absorbent sample pad will preferably becomplementary in shape for the receiving port such that when theabsorbent sample pad is inserted into the receiving port and contactsthe absorbent membrane, the analytical test device becomes substantiallyfluid-tight.

[0064] As used herein, the term “receiving port” refers to an aperturein the top, or the bottom or optionally an aperture in the top and thebottom together that allows the absorbent sample pad to enter theassembled top and bottom and contact the absorbent membrane. Preferably,the receiving port will be complementary in size to the absorbent samplepad to ensure a tight fit.

[0065] As used herein the term “container” refers to a material capableof supporting the absorbent sample pad. The container is preferably madeof any suitable material that prevents the user from coming into directcontact with the fluid sample, which would otherwise contaminate theassay and make its use unpleasant. Specifically, the container in oneembodiment of the invention is made of a non-porous material such as thecommercially available “ABS” plastic (Monsanto Co., St. Louis, Mo.)comprising a top and a bottom (see FIGS. 1, 2, 3 and 4). It is alsounderstood that the container could be in the shape of a stick, rod ortongue depressor.

[0066] Also, as used herein the term “stopping means” refers to astructure capable of stopping the movement of the container so that theabsorbent sample pad is in direct fluid flow contact with the absorbentmembrane but does not allow the absorbent sample pad to come into directfluid flow contact with the reaction membrane. In one embodiment, theassembled top and bottom are the stopping means for the container. Inanother embodiment a horizontal bar on the bottom is the stopping meansfor the container.

[0067] The terms “absorbent membrane” and “reaction membrane” refer toany bibulous, porous or fibrous material capable of rapidly absorbing anaqueous fluid and conducting the fluid via capillary attraction.Suitable materials are described, for example, in H. J. Friesen, U.S.Pat. No. 4,861,711, issued Aug. 29, 1989; J. Bunting, U.S. Pat. No.4,271,140, issued Jun. 2, 1981; May et al., U.S. Pat. No. 5,622,871,issued Apr. 22, 1997; May et al., U.S. Pat. No. 5,656,503, issued Aug.12, 1997; May et al., U.S. Pat. No. 5,602,040, issued Feb. 11, 1997; andR. Rosenstein, U.S. Pat. No. 5,591,645, issued Jan. 7, 1997 (each ofwhich is incorporated by reference herein). The preferred material forthe absorbent membrane is a fiberglass product such as that marketed as“MANNIWEB” or “MANNIGLAS” by Lydall, Inc., Manchester, Conn. Othersuitable materials include polyethylene or nitrocellulose pads andstrips. The means for binding reagents to these materials are well-knownin the art. Preferred porous materials include nitrocellulose, nylon,paper and silica gel. An advantage of a nitrocellulose membrane is thatan immobilized antibody described above can be attached without priorchemical treatment. However, antibodies can be immobilized on othermaterials such as filter paper using well known chemical couplingmethods such as, for example, CNBr, carbonyldimidazole or tresylchloride. The reaction membrane will preferably be a chromatographicstrip coated with gelatin to enhance the life of the strip and clarityof any visible reactions produced in the test.

[0068] The present invention also provides methods of using theanalytical test device for detecting an analyte in a fluid sample,comprising adding fluid sample to the absorbent sample pad of theanalytical test device, contacting the absorbent sample pad with theabsorbent membrane and observing the reaction complex through thedisplay port. Preferably, the reaction complex will be visible. Alsopreferably, there will be a first display port and a second displayport.

[0069]FIG. 1 shows a perspective view of one embodiment of an analyticaltest device for performing assays in accord with the invention. FIG. 1,therefore, depicts a housing 106 of rectangular shape, although anyappropriate shape may be employed. Housing 106 is composed of two parts.The first is top 100, in which one opening appears, and the second isbottom 112. The opening through housing top 100 is display port 102through which the test result can be viewed and compared. An openingthrough bottom 112 is receiving port 118. Absorbent sample pad 108 is incontainer 126. Container 126 is preferably composed of two parts, top128 and bottom 130.

[0070]FIG. 2 is an exploded view of the analytical test device ofFIG. 1. In this view, it can be seen that bottom 112 contains floor 143,sidewalls 145, snaps 128 and receiving port 118. Contained withinhousing 106 will be membranes containing the reagents for carrying outthe assay. In one embodiment there will be absorbent membrane 124 andreaction membrane 122. Absorbent membrane 124 and reaction membrane 122will be in contact with, but need not be affixed, to each other. Inaddition, they will be disposed along floor 143 in the assembled devicewith absorbent membrane 124 toward receiving port 118 and reactionmembrane 122 beneath display port 102. Absorbent sample pad 108 forms aseparate part of the device which can be contacted with fluid sample,and then inserted through receiving port 118 to be in fluid flow contactwith absorbent membrane 124.

[0071]FIG. 3 shows a perspective view of another embodiment of theanalytical test device for performing assays in accord with theinvention. To that end housing 207 is composed of top 208, bottom 216,and has a first display port 210 and second display port 212 positionedon top 208, preferably in close proximity for ease of comparison ofresults. In addition, housing 208 has receiving port 214. Insidecontainer 202, will be absorbent sample pad 204. Container 202 ispreferably composed of top 200 and bottom 206.

[0072]FIG. 4 is an exploded view of the analytical test device shown inFIG. 3, and shows the components, top 208, bottom 216, lower plane 221,upper plane 240, slope 234, sidewalls 223, snaps 218, locating points232, 242, 228 and 238, fluid gully 224, receiving port 214, andmembranes containing the reagents for carrying out the assay, asdescribed below. To that end, it can be seen that contained withinhousing 207 is absorbent membrane 224, reaction membrane 222 andcollecting pad 220. Absorbent membrane 224 will be placed along lowerplane 221 and along slope 234. Preferably, absorbent membrane 224 willbe affixed to a nonporous substrate 226 such as vinyl coated paper orplastic coated paper by an adhesive or other suitable means. Substrate226 will extend along upper plane 240 to form a surface to whichreaction membrane 222 will be affixed. Absorbent membrane 224 andreaction membrane 222 will be in contact with, but need not be affixed,to each other. In another embodiment, absorbent membrane 224 will besplit at its point of attachment to substrate 226 to extend above andbelow the substrate. This configuration serves to regulate the flow offluid sample from absorbent membrane 224 to reaction membrane 222. Flowcontrol can also be enhanced by placement of a well or fluid gully 224below the junction between membranes 224 and 222. FIG. 4 also shows theplacement of absorbent sample pad 204 in container 202 between top 200and bottom 206, which are preferably held together by snaps 218. Inaddition, ridges 236 hold absorbent sample pad 204 in place.

[0073]FIG. 5 is an enlarged sectional view along line 3-3 of theanalytical test device shown in FIG. 3. In this view the orientation ofabsorbent sample pad 204 can be seen. Vertical bar 250 can be seenholding absorbent membrane 224 down to follow the contour of slope 234.Absorbent sample pad 204 is not in fluid flow contact with absorbentmembrane 224 at this point. Thus, the user has the opportunity to addadditional samples or reagents if desired.

[0074]FIG. 6 is another enlarged sectional view along line 3-3 of theanalytical test device shown in FIG. 3. In this view absorbent samplepad 204 is in fluid flow contact with absorbent membrane 224 afterhaving been contacted with fluid sample, and then inserted intoreceiving port 214. Contact with absorbent membrane 324 will beindicated by a resistance. In this embodiment of the analytical testdevice, assembled top 208 and bottom 216 serve as stopping means forabsorbent sample pad 204. Vertical bar 250 assures that absorbentmembrane 224 is held down so that it can contact absorbent sample pad204.

[0075]FIG. 7 shows a perspective view of another embodiment of theanalytical test device. To that end, housing 306 is composed of top 300,bottom 312 and has first display port 304 and second display port 302positioned on top 300, preferably in close proximity for ease ofcomparison of results. Housing 306 also has knob 314 that can be slidalong groove 316 to stop point 315. Knob 314 is connected to andcontrols the movement of container 310 containing absorbent sample pad308.

[0076]FIG. 8 is an exploded view of the analytical test device shown inFIG. 7 and shows the components, bottom 312, sidewalls 336, snaps 328,slope 342, upper plane 346, lower plane 343, receiving port 318,stopping means 332, locating points 334, 336, 338 and 340, fluid gully330 and membranes containing the reagents for carrying out the assay, asdescribed below. To that end it can be seen that contained withinhousing 306 is absorbent membrane 324, reaction membrane 322 andcollecting pad 320. Absorbent membrane 324 will be placed along lowerplane 343 and along slope 342. Preferably, absorbent membrane 324 willbe affixed to a nonporous substrate 326 such as vinyl coated paper orplastic coated paper by an adhesive or other suitable means. Substrate326 will extend along upper plane 346 to form a surface to whichreaction membrane 322 will be affixed. Absorbent membrane 324 andreaction membrane 322 will be in contact with, but need not be affixed,to each other. In another embodiment, absorbent membrane 324 will besplit at its point of attachment to substrate 326 to extend above andbelow the substrate. This configuration serves to regulate flow of fluidsample from absorbent membrane 324 to reaction membrane 322. Flowcontrol can also be enhanced by placement of a well or fluid gully 330below the junction between membranes 324 and 322.

[0077]FIG. 9 is an enlarged sectional view along line 3-3 of theanalytical test device shown in FIG. 7. In this view the orientation ofabsorbent sample container 310, absorbent sample pad 308 and absorbentmembrane 324 can be seen. Vertical bar 350 extends downward from the topand holds absorbent membrane 324 along slope 342. Stopping means 332 canalso be seen. Fluid sample is added to absorbent sample pad 308. It canbe seen that absorbent sample pad 308 is not in fluid flow contact withabsorbent membrane 324 at this point. Thus, the user has the opportunityto add additional samples or reagents if desired.

[0078]FIG. 10 is another enlarged sectional view along line 3-3 of theanalytical test device shown in FIG. 7. It can be seen that absorbentsample pad 308 is in fluid flow contact with absorbent membrane 324 atthis point, having been stopped by stopping means 332.

[0079] In FIGS. 11, 12 and 13, analyte in the fluid sample, if present,will bind to the diffusively bound labeled reagent at point 425 onabsorbent membrane 424 to form a first complex. If desired, furtherfluid sample may be added up to the maximum binding capacity ofunlabeled reagent and absorbency of the collecting pad material 420. Thefirst complex and unbound first reagent, if any, will be carried alongwith the fluid sample by capillary action up to the reaction membrane422. The position of the non-diffusively bound labeled reagent and firstcomplex, if any, are indicated as 425 on FIGS. 11, 12 and 13.

[0080] All first complexes carried with the fluid sample will contactthe reaction membrane 422. Fluid sample will pass through the reactionmembrane 422, bringing the first complex, if any, into contact with thenon-diffusively bound reagent to the reaction membrane 422 to bindtherewith to form a second complex composed of firstcomplex/non-diffusively bound reagent. If second complexes are formed,they will appear in the test region 427 (FIG. 12).

[0081] Fluid sample will continue its migration by wicking throughreaction membrane 422 past the non-diffusively bound reagent to thenon-diffusively bound control reagent. Diffusively bound labeled reagentnot complexed to analyte will bind with the non-diffusively boundcontrol reagent to form a third complex. The third complex will appearas display 421 in the control region (FIGS. 11, 12 and 13). Substrate426 is also shown.

[0082] Formation of all complexes in the assay method described hereinmay be by sandwich or competitive immunoassay techniques well known tothose skilled in the art. Within a predetermined time period, any secondcomplex present and the third complex will produce a detectable signalmediated by the label. In one embodiment, the detectable signal will bea color change. This color change will signal either a positive (analytepresent) or negative (analyte not present) reaction by providing twoseparate reactive regions within those portions of reaction membrane 422visible to the user through the display ports.

[0083] Other control or comparative result signals may be provided,including signals that indicate whether an invalid result is obtained,by similar means known to those skilled in the art (see, for example,the signal system described in Brown et al., U.S. Pat. No. 5,160,701,issued Nov. 3, 1992, which is incorporated by reference herein).

[0084] As shown in FIG. 11, a positive result is indicated when colorchanges forming substantially similar shapes (in this instance,horizontal bars 421 and 423) appear. In contrast, as shown in FIG. 12, anegative result is indicated when a color change forming adistinguishable shape only appears, for example, as a horizontal bar421. Finally, an inconclusive result is shown in FIG. 13, where colorchanges appear in both regions having substantially dissimilar shapes(i.e., horizontal bar 421 and smudge 429). In this latter instance, theuser would be alerted that the test should be repeated. While due to thedecreased impact of leading edge effects and similar phenomena in thisassay it is not expected that inconclusive results will be frequent, theassay requires such a small application of fluid sample that repetitionshould not be problematic in most applications.

[0085]FIG. 14 and FIG. 15 show perspective views of the absorbent andreaction membranes wherein a competitive assay is employed. FIG. 14shows a positive assay result, as indicated by no signal in the displayport. FIG. 15 shows a negative assay result, as indicated by thepresence of bar 452 in the display port.

EXAMPLE I Analytical Test Device with Separate Absorbent Sample Pad

[0086] This example describes the method and analytical test devicedepicted in FIG. 3 containing a separate absorbent sample pad that isinserted into the receiving port.

[0087] This assay procedure is performed using anti-hCG antibody to formthe second complex and an anti-hCG-colloidal gold compound to form thefirst complex. A fluid sample can be collected at any time of day, butfor best results, it is best to test the first urine of the morningbecause it contains the highest concentration of hCG. The fluid sampleis 1 cubic centimeter (hereinafter “cc”) of urine suspected ofcontaining hCG; the assay is performed at ambient temperatures not below15° C. or exceeding 30° C. Absorbent sample pad 204 is placed intocontact with a sample of urine, either by dipping it into a containercontaining at least 1 cc of urine, or by holding container 202containing absorbent sample pad 204 into a stream of urine. Absorbentsample pad 204 is then inserted into receiving port 214 so thatabsorbent sample pad contacts absorbent membrane 224. Within 3 minutesvisible color changes, (from pure white to pink) through substantiallythe center of display ports 210 and 212 on reaction membrane 222, arefaintly visible. By the end of five minutes, a vividly pink bar symbolhas appeared through substantially the center of each display port,indicating the presence of hCG in the fluid sample. All other visibleareas of the reaction membrane visible through the second display portremain white, which is the normally visible color of the reactionmembrane. This assay may be performed at any time after a suspectedconception to determine the presence of hCG and, therefore, pregnancy.

EXAMPLE II Analytical Test Device with Container Disposed Between Topand Bottom of Device

[0088] This example describes the method and analytical test devicedepicted in FIG. 7 containing a absorbent sample pad and containerdisposed between the top and bottom of the device.

[0089] The assay procedure is performed using anti-hCG antibody to formthe second complex and an anti-hCG-colloidal gold compound to form thefirst complex. A fluid sample can be collected at any time of day, butfor best results, it is best to test the first urine of the morningbecause it contains the highest concentration of hCG. The fluid sampleis at least 1 cubic centimeter (hereinafter “cc”) of urine suspected ofcontaining hCG; the assay is performed at ambient temperatures not below15° C. or exceeding 30° C. The 1 cc of urine is added to absorbentsample pad 308 by pipette. The container 310 is slid using knob 314along groove 316 to its stop position 315. Within 3 minutes visiblecolor changes, (from pure white to pink) through substantially thecenter of display ports 304 and 302 on reaction membrane 322, arefaintly visible. By the end of five minutes, a vividly pink bar symbolhas appeared through substantially the center of each display port,indicating the presence of hCG in the sample. All other visible areas ofthe reaction membrane visible through the second display port remainwhite, which is the normally visible color of the reaction membrane.This assay may be performed at any time after a suspected conception todetermine the presence of hCG and, therefore, pregnancy.

[0090] Although the invention has been described with reference to theexamples provided above, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the claims.

I claim:
 1. An analytical test device, comprising: (a) a top having oneor more display ports and, optionally, a receiving port and a verticalbar; (b) a bottom, optionally having the receiving port, an upper plane,a slope and a lower plane; (c) a strip comprising an absorbent membrane,a reaction membrane having one or more reagents that form a reactioncomplex with an analyte in a fluid sample and, optionally, a collectingpad; (d) an absorbent sample pad in a container; and (e) a stoppingmeans, wherein the absorbent membrane, the reaction membrane and thecollecting pad, when present, are in fluid flow contact with oneanother; the strip is placed on the bottom such that the reactionmembrane is visible through the display port and the absorbent membraneis towards the receiving port; the top or the bottom have the receivingport or together the top and the bottom form the receiving port; and inthe assembled top and bottom the absorbent sample pad is slidablyinsertable through the receiving port until stopped by the stoppingmeans, which places the absorbent sample pad in fluid flow contact withthe absorbent membrane.
 2. The analytical test device of claim 1,further comprising the top having the optional vertical bar, wherein thevertical bar deflects the absorbent membrane to follow the contour ofthe slope and further comprising the bottom having the optional upperplane, slope and lower plane.
 3. The analytical test device of claim 1,wherein the stopping means is the container contacting the outside ofthe assembled top and bottom.
 4. The analytical test device of claim 1,further comprising a bottom having a horizontal bar, wherein thehorizontal bar is inside the assembled top and bottom and is thestopping means.
 5. The analytical test device of claim 4, wherein thestopping means is the horizontal bar contacting the container.
 6. Theanalytical test device of claim 1, wherein the edge of the absorbentsample pad that contacts the absorbent membrane is orthogonal to theabsorbent membrane.
 7. The analytical test device of claim 1, whereinthe one or more reagents is a diffusively bound labeled reagent bound tothe absorbent membrane at a point spatially separated from the point atwhich the absorbent sample pad first contacts the absorbent membrane. 8.The analytical test device of claim 7, wherein bound to the reactionmembrane is: (a) a non-diffusively bound reagent complementary to ananalyte in the fluid sample at a point beneath the display port; and (b)a non-diffusively bound control reagent complementary to the diffusivelybound labeled reagent at a point beneath the display port spatiallyseparated from the point at which the non-diffusively bound reagent isbound.
 9. The analytical test device according to claim 8, furthercomprising a first display port and a second display port, wherein thenon-diffusively bound reagent is positioned beneath the first displayport and the non-diffusively bound control reagent is positioned beneaththe second display port.
 10. The analytical test device of claim 7,wherein bound to the reaction membrane at a point beneath the displayport is a non-diffusively bound reagent, wherein the analyte and thediffusively bound labeled reagent compete for binding to thenon-diffusively bound reagent.
 11. A method for detecting an analyte ina fluid sample, comprising the steps of: (a) adding fluid sample to theabsorbent sample pad of the analytical test device of claim 1; (b)inserting the absorbent sample pad into the receiving port until theabsorbent sample pad is stopped by the stopping means; (c) detecting theanalyte by observing the reaction complex through the display port. 12.The method according to claim 11, wherein the reaction complex isvisible.